Techniques in molecular biology and molecular medicine often rely on analysis of single biological molecules. Such techniques include DNA and RNA sequencing, polymorphism detection, the detection of proteins of interest, the detection of protein-nucleic acid complexes, and many others. The high sensitivity, high throughput and low reagent costs involved in single molecule analysis make this type of analysis an increasingly attractive approach for a variety of detection and analysis problems in molecular medicine, from low cost genomics to high sensitivity marker analysis.
The small observation volumes often used for single molecule analysis methods are typically provided by immobilizing or otherwise localizing molecules of interest within an optical confinement reaction/observation region, such as an array of extremely small wells as in an array of Zero Mode Waveguides (ZMWs), and delivering molecules of interest (including for example, a template, primers, enzymes etc.,) to the reaction region. One difficulty in performing single molecule analyses occurs in loading the reaction/observation region of single molecule analysis devices with the molecules of interest (e.g., template or other analyte and/or enzyme). Entropic barriers to loading can be significant when attempting to load large reactant molecules into these nanoscale reaction sites.
While passive distribution methods are effective in ensuring that, in most cases, not more than a single template or enzyme (or other analyte) molecule is loaded in each observation/reaction volume in an array such as a ZMW array, it would be desirable to develop methods and compositions for increasing the efficiency and density of loading in such reaction sites. The present invention provides these and other features that will be apparent upon complete review of the following.